Methods and materials for assessing allelic imbalance

ABSTRACT

Methods and systems for detecting allelic imbalance using nucleic acid sequencing are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/412,404, filed Jan. 23, 2017, which is a continuation of U.S.application Ser. No. 15/010,721, filed Jan. 29, 2016 (issued as U.S.Pat. No. 9,574,229), which is a continuation of U.S. application Ser.No. 14/109,163, filed Dec. 17, 2013 (issued as U.S. Pat. No. 9,279,156),which is a continuation of International Application No.PCT/US12/042668, filed Jun. 15, 2012, which claims the priority benefitof U.S. Provisional Application No. 61/498,418, filed Jun. 17, 2011, theentire contents of each of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention generally relates to molecular diagnosis, and particularlyto a method and system for detecting allelic imbalance in patientsamples.

BACKGROUND OF THE INVENTION

In general, a comparison of sequences present at the same locus on eachchromosome (each autosomal chromosome for males) of a chromosome paircan reveal whether that particular locus is homozygous or heterozygouswithin the genome of a cell. Polymorphic loci within the human genomeare generally heterozygous within an individual since that individualtypically receives one copy from the biological father and one copy fromthe biological mother. In some cases, a polymorphic locus or a string ofpolymorphic loci within an individual are homozygous as a result ininheriting identical copies from both biological parents. In othercases, homozygosity results from a loss of heterozygosity (LOH) from thegermline. Because LOH and copy number information can be clinicallyuseful, there is a need for improved methods of identifying loci andregions of LOH in samples.

BRIEF SUMMARY OF THE INVENTION

Copy number (including allelic imbalance and LOH) analysis of tumortissues has been traditionally performed using single nucleotidepolymorphism (SNP) arrays. The data quality is often highly variableand, especially for FFPE samples, tends to be poor. The inventors havedeveloped a method of genome-wide copy number analysis that produceshigh quality data from all sample types that is based on in-solutioncapture of DNA fragments spanning target loci (e.g., SNPs), followed byparallel sequencing to identify and quantitate the alleles. Theresulting data allows high quality LOH and copy number analysis of thesample.

Accordingly, in one aspect of the present invention, a method ofdetecting allelic imbalance status in a plurality of genomic loci in atumor sample from a cancer patient is provided, comprising the steps ofenriching a genomic DNA sample for DNA molecules each comprising a locusof interest; sequencing said DNA molecules to determine the genotype ateach such locus; determining for each locus whether there is allelicimbalance.

In another aspect of the present invention, a method of detecting LOHstatus in a plurality of genomic loci in a tumor sample from a cancerpatient is provided, comprising the steps of enriching a genomic DNAsample for DNA molecules each comprising a locus of interest; sequencingsaid DNA molecules to determine the genotype at each such locus;determining for each homozygous locus whether it is homozygous due toLOH.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting allele dosages of breast cancer cells from abreast cancer patient along chromosome 1 as determined using a SNParray. The chromosome region between the arrows is an LOH region that isabout 103 Mb in length.

FIG. 2 is a graph plotting allele dosages of breast cancer cells for thesame breast cancer patient as on FIG. 1 along chromosome 1 as determinedusing high-throughput sequencing. The chromosome region between thearrows is an LOH region that is about 103 Mb in length.

FIG. 3 is a diagram of an example of a computer device and a mobilecomputer device that can be used to implement the techniques describedherein.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly discovered that determining allelic imbalance(e.g., abnormal copy number, LOH) in formalin-fixed paraffin-embedded(“FFPE”) samples using sequencing of genomic regions comprising loci ofinterest (e.g., SNPs) yields far superior quality data when compared tocopy number and allelic imbalance data generated using microarrays. Thisinvention enables large-scale (e.g., whole genome) copy number (e.g.,allelic imbalance) analysis of samples of varying quality. Inparticular, it enables high quality data to be produced fromFFPE-derived DNA. Current array-based platforms are unable to producedata of sufficient quality from this sample type.

Accordingly, in one aspect of the present invention, a method ofdetecting allelic imbalance status in a plurality of genomic loci in atumor sample from a cancer patient is provided, comprising the steps ofenriching a genomic DNA sample for DNA molecules each comprising a locusof interest; sequencing said DNA molecules to determine the genotype ateach such locus; determining for each locus whether there is allelicimbalance. “Locus” as used herein has its usual meaning in the art. Asused herein, “region” means a plurality of substantially adjacent loci.Unless stated otherwise or unless the context clearly indicatesotherwise, statements made about a locus will generally apply to aregion.

As used herein, “allelic imbalance” means any instance where the somaticcopy number differs from the germline copy number at a genomic locus orregion. In some embodiments allelic imbalance is expressed in terms ofmajor copy proportion (“MCP”). Major copy proportion and MCP, as usedherein, mean the ratio of the major allele copy number to themajor+minor allele copy number, as follows:MCP=[major allele copy number]/([major allele copy number]+[minor allelecopy number])In some embodiments, a locus or region shows allelic imbalance if theMCP at such locus or region is 0.51, 0.52, 0.53, 0.54, 0.55, 0.6, 0.65,0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.

One example of allelic imbalance is loss of heterozygosity (“LOH”), inwhich a locus is heterozygous in the germline but homozygous in somatictissue. In this sense, homozygosity can include homozygous loss (i.e.,deletion) of the locus in somatic tissue. The different types ofpossible LOH and allelic imbalance are discussed in more detail below.

Thus in some embodiments the present invention provides a method ofdetecting LOH status in a plurality of genomic loci in a tumor samplefrom a cancer patient, comprising enriching a genomic DNA sample for DNAmolecules each comprising a locus of interest; sequencing said DNAmolecules to determine the genotype at each such locus; determining foreach homozygous locus whether it is homozygous due to LOH.

According to the present invention, nucleic acid sequencing techniquescan be used to identify loci and/or regions as having allelic imbalance.For example, genomic DNA from a cell sample (e.g., a cancer cell sample)can be extracted and fragmented. Any appropriate method can be used toextract and fragment genomic nucleic acid including, without limitation,commercial kits such as QIAamp DNA Mini Kit (Qiagen), MagNA Pure DNAIsolation Kit (Roche Applied Science) and GenElute Mammalian Genomic DNAMiniprep Kit (Sigma-Aldrich). Once extracted and fragmented, eithertargeted or untargeted sequencing can be done to determine the sample'sgenotypes at loci of interest. For example, whole genome, wholetranscriptome, or whole exome sequencing can be done to determinegenotypes at millions or even billions of base pairs (i.e., base pairscan be “loci” to be evaluated).

In some cases, targeted sequencing of known polymorphic loci (e.g., SNPsand surrounding sequences) can be done as an alternative to microarrayanalysis. For example, the genomic DNA can be enriched for thosefragments containing a locus (e.g., SNP location) to be analyzed usingkits designed for this purpose (e.g., Agilent SureSelect, IlluminaTruSeq Capture, Nimblegen SeqCap EZ Choice, Raindance Thunderstorm™).For example, genomic DNA containing the loci to be analyzed can behybridized to biotinylated capture RNA fragments to form biotinylatedRNA/genomic DNA complexes. Alternatively, DNA capture probes may beutilized resulting in the formation of biotinylated DNA/genomic DNAhybrids. Streptavidin coated magnetic beads and a magnetic force can beused to separate the biotinylated RNA/genomic DNA complexes from thosegenomic DNA fragments not present within a biotinylated RNA/genomic DNAcomplex. The obtained biotinylated RNA/genomic DNA complexes can betreated to remove the captured RNA from the magnetic beads, therebyleaving intact genomic DNA fragments containing a locus to be analyzed.These intact genomic DNA fragments containing the loci to be analyzedcan be amplified using, for example, PCR techniques. Alternatively, amultiplex PCR reaction can be employed to enrich for loci of interest.PCR primers can be designed to flank loci of interest and a PCR reactioncan be run to amplify sequences comprising such loci.

The enriched genomic DNA fragments can be sequenced using any sequencingtechnique. Beyond Sanger sequencing, numerous suitable sequencingmachines and strategies are well known in the art, including but notlimited to those developed by IIlumina (the Genome Analyzer; Bennett etal. (2005) Pharmacogenomics, 6:373-382; HiSeq; MiSeq); by AppliedBiosystems, Inc. (the SOLiD™ Sequencer; solid.appliedbiosystems.com); byRoche (e.g., the 454 GS FLX™ sequencer; Margulies et al. (2005) Nature,437:376-380; U.S. Pat. Nos. 6,274,320; 6,258,568; 6,210,891); by HelicosBiosciences (Heliscope™ system, see, e.g., U.S. Patent App. Pub. No.2007/0070349); by Oxford Nanopore (e.g., GridION™ and MinION™, see,e.g., International Application No. PCT/GB2009/001690, pub. no.WO/2010/004273); and by others.

The sequencing results from the genomic DNA fragments can be used toidentify loci as having allelic imbalance. In some cases, an analysis ofthe allelic imbalance status of loci over a length of a chromosome canbe performed to determine the length of regions of allelic imbalance.For example, a stretch of SNP locations that are spaced apart (e.g.,spaced about 25 kb to about 100 kb apart) along a chromosome can beevaluated by sequencing, and the sequencing results used to determinenot only the presence of a region of allelic imbalance ( ) e.g., somatichomozygosity) along a chromosome but also the length of that region ofimbalance. Obtained sequencing results can be used to generate a graphthat plots allele dosages along a chromosome. Allele dosage d_(i) forSNP i can be calculated from the adjusted number of captured probes fortwo alleles (A_(i) and B_(i)): d_(i)=.A_(i)/(A_(i)+B_(i)). An example ofsuch a graph is presented in FIG. 2.

Once a sample's genotype (e.g., homozygosity) has been determined for aplurality of loci (e.g., SNPs), common techniques can be used toidentify loci and regions of allelic imbalance due to somatic change(e.g., LOH). One way to determine whether imbalance is due to somaticchange is to compare the somatic genotype to the germline. For example,the genotype for a plurality of loci (e.g., SNPs) can be determined inboth a germline (e.g., blood) sample and a somatic (e.g., tumor) sample.The genotypes for each sample can be compared (typicallycomputationally) to determine where the genome of the germline cell was,e.g., heterozygous and the genome of the somatic cell is, e.g.,homozygous. Such loci are LOH loci and regions of such loci are LOHregions.

Computational techniques can also be used to determine whether allelicimbalance is somatic (e.g., due to LOH). Such techniques areparticularly useful when a germline sample is not available for analysisand comparison. For example, algorithms such as those describedelsewhere can be used to detect allelic imbalance regions usinginformation from SNP arrays (Nannya et al., Cancer Res., 65:6071-6079(2005)). Typically these algorithms do not explicitly take into accountcontamination of tumor samples with benign tissue. Cf. InternationalApplication No. PCT/US2011/026098 to Abkevich et al.; Goransson et al.,PLoS One (2009) 4(6):e6057. This contamination is often high enough tomake the detection of allelic imbalance regions challenging. Improvedanalytical methods according to the present invention for identifyingallelic imbalance, even in spite of contamination, include thoseembodied in computer software products as described below.

The following is one example. If the observed ratio (e.g., MCP) of thesignals of two alleles, A and B, is two to one, there are twopossibilities. The first possibility is that cancer cells have LOH withdeletion of allele B in a sample with 50% contamination with normalcells. The second possibility is that there is no LOH but allele A isduplicated in a sample with no contamination with normal cells. Analgorithm can be implemented as a computer program as described hereinto reconstruct LOH regions based on genotype (e.g., SNP genotype) data.One point of the algorithm is to first reconstruct allele specific copynumbers (ASCN) at each locus (e.g., SNP). ASCNs are the numbers ofcopies of both paternal and maternal alleles. An LOH region is thendetermined as a stretch of SNPs with one of the ASCNs (paternal ormaternal) being zero. The algorithm can be based on maximizing alikelihood function and can be conceptually akin to a previouslydescribed algorithm designed to reconstruct total copy number (ratherthan ASCN) at each locus (e.g., SNP). See International Application No.PCT/US2011/026098 (pub. no. WO/2011/106541) (hereby incorporated byreference in its entirety). The likelihood function can be maximizedover ASCN of all loci, level of contamination with benign tissue, totalcopy number averaged over the whole genome, and sample specific noiselevel. The input data for the algorithm can include or consist of (1)sample-specific normalized signal intensities for both allele of eachlocus and (2) assay-specific (specific for different SNP arrays and forsequence based approach) set of parameters defined based on analysis oflarge number of samples with known ASCN profiles.

In some cases, a selection process can be used to select loci (e.g., SNPloci) to be evaluated using an assay configured to identify loci ashaving allelic imbalance (e.g., SNP array-based assays andsequencing-based assays). For example, any human SNP location can beselected for inclusion in a SNP array-based assay or a sequencing-basedassay configured to identify loci as having allelic imbalance within thegenome of cells. In some cases, 0.5, 1.0, 1.5, 2.0, 2.5 million or moreloci (e.g., SNP locations) present within the human genome can beevaluated to identify those loci that (a) are not present on the Ychromosome, (b) are not mitochondrial loci, (c) have a minor allelefrequency of at least about 5% in the population of interest (e.g.,Caucasians), (d) have a minor allele frequency of at least about 1% inthree populations other than the population of interest (e.g., Chinese,Japanese, and Yoruba), and/or (e) do not have a significant deviationfrom Hardy-Weinberg equilibrium in any of these populations. In somecases, more than 100,000, 150,000, or 200,000 human loci can be selectedthat meet criteria (a) through (e). Of the human loci meeting criteria(a) through (e), a group of loci (e.g., top 2,500, 5,000, 7,500, 10,000,20,000, 30,000, 40,000, 50,000, 75,000, 100,000, 150,000, or 200,000loci) can be selected such that the loci have a high degree of allelefrequency in the population of interest, cover the human genome in asomewhat evenly spaced manner (e.g., at least one locus of interestevery about 5 kb, 10 kb, 25 kb, 50 kb, 75 kb, 100 kb, 150 kb, 200 kb,300 kb, 400 kb, 500 kb or more), and are not in linkage disequilibriumwith another selected locus in any of the populations used for analysis.In some cases, about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 thousandor more loci can be selected as meeting each of these criteria andincluded in an assay configured to identify allelic imbalance regionsacross a human genome. For example, between about 70,000 and about90,000 (e.g., about 80,000) SNPs can be selected for analysis with a SNParray-based assay, and between about 45,000 and about 55,000 (e.g.,about 54,000) SNPs can be selected for analysis with a sequencing-basedassay.

Accordingly, in one aspect of the present invention, a method ofdetecting allelic imbalance status in a plurality of genomic loci in asample from a patient is provided, comprising the steps of enriching agenomic DNA sample for DNA molecules each comprising a locus ofinterest; sequencing said DNA molecules to determine the genotype ateach such locus; determining for each locus whether it has allelicimbalance.

In another aspect of the present invention, a method of detecting LOHstatus in a plurality of genomic loci in a sample from a patient isprovided, comprising the steps of enriching a genomic DNA sample for DNAmolecules each comprising a locus of interest; sequencing said DNAmolecules to determine the genotype at each such locus; determining foreach homozygous locus whether it is homozygous due to LOH.

In another aspect of the present invention, a method of detecting copynumber status in a plurality of genomic loci in a sample from a patientis provided, comprising the steps of enriching a genomic DNA sample forDNA molecules each comprising a locus of interest; sequencing said DNAmolecules; and quantitating each allele at each such locus to determineits copy number.

In some embodiments at least 10, 50, 100, 1,000, 10,000, 50,000, 55,000,75,000, 100,000, 150,000, 200,000, 300,000, 400,000, 500,000, 750,000,1,000,000, 2,000,000 or more loci are evaluated. In some embodimentsthese loci are spaced evenly along the genome. As used herein, loci are“evenly spaced along the genome” when the percentage difference betweenthe distance_(AB) between any two loci A and B and the distance_(CD)between any other two loci C and D (i.e.,100*(distance_(AB)−distance_(CD))/distance_(AB) or100*(distance_(AB)−distance_(CD))/distance_(CD)) is less than or equalto 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.Such percentage difference is referred to herein as the “genomicspacing” of loci. In some embodiments the sample is an FFPE tissuesample. In some embodiments the sample is a tumor sample from thepatient.

Another aspect of the invention provides a system for determiningallelic imbalance status in a plurality of loci in a sample comprising:a sample analyzer for (1) enriching a genomic DNA sample for DNAmolecules each comprising a locus of interest and (2) sequencing saidDNA molecules to produce a plurality of quantitative signals about eachsuch locus; a computer program for analyzing said plurality ofquantitative signals to determine whether each such locus has allelicimbalance.

Another aspect of the invention provides a system for determining LOHstatus in a plurality of loci in a sample comprising: a sample analyzerfor (1) enriching a genomic DNA sample for DNA molecules each comprisinga locus of interest and (2) sequencing said DNA molecules to produce aplurality of quantitative signals about each such locus; a computerprogram for analyzing said plurality of quantitative signals todetermine whether each such locus is homozygous in the sample; and acomputer program for determining for each homozygous locus whether it ishomozygous due to LOH.

Another aspect of the invention provides a system for detecting copynumber status in a plurality of genomic loci in a sample from a patientcomprising: a sample analyzer for (1) enriching a genomic DNA sample forDNA molecules each comprising a locus of interest and (2) sequencingsaid DNA molecules to produce a plurality of quantitative signals abouteach such locus; and a computer program for analyzing said plurality ofquantitative signals to quantitate each allele at each such locus todetermine its copy number.

In some embodiments of the systems of the invention, one sample analyzerboth enriches the sample for DNA of interest and sequences that DNA. Inother embodiments two or more sample analyzers perform these functions.In some embodiments, one software program analyzes the plurality ofquantitative signals to determine whether each locus is homozygous inthe sample and also determines for each homozygous locus whether it ishomozygous due to LOH.

FIG. 3 is a diagram of an example of a computer device 1400 and a mobilecomputer device 1450, which may be used with the techniques describedherein. Computing device 1400 is intended to represent various forms ofdigital computers, such as laptops, desktops, workstations, personaldigital assistants, servers, blade servers, mainframes, and otherappropriate computers. Computing device 1450 is intended to representvarious forms of mobile devices, such as personal digital assistants,cellular telephones, smart phones, and other similar computing devices.The components shown here, their connections and relationships, andtheir functions, are meant to be exemplary only, and are not meant tolimit implementations of the inventions described and/or claimed in thisdocument.

Computing device 1400 includes a processor 1402, memory 1404, a storagedevice 1406, a high-speed interface 1408 connecting to memory 1404 andhigh-speed expansion ports 1410, and a low speed interface 1415connecting to low speed bus 1414 and storage device 1406. Each of thecomponents 1402, 1404, 1406, 1408, 1410, and 1415, are interconnectedusing various busses, and may be mounted on a common motherboard or inother manners as appropriate. The processor 1402 can processinstructions for execution within the computing device 1400, includinginstructions stored in the memory 1404 or on the storage device 1406 todisplay graphical information for a GUI on an external input/outputdevice, such as display 1416 coupled to high speed interface 1408. Inother implementations, multiple processors and/or multiple buses may beused, as appropriate, along with multiple memories and types of memory.Also, multiple computing devices 1400 may be connected, with each deviceproviding portions of the necessary operations (e.g., as a server bank,a group of blade servers, or a multi-processor system).

The memory 1404 stores information within the computing device 1400. Inone implementation, the memory 1404 is a volatile memory unit or units.In another implementation, the memory 1404 is a non-volatile memory unitor units. The memory 1404 may also be another form of computer-readablemedium, such as a magnetic or optical disk.

The storage device 1406 is capable of providing mass storage for thecomputing device 1400. In one implementation, the storage device 1406may be or contain a computer-readable medium, such as a floppy diskdevice, a hard disk device, an optical disk device, or a tape device, aflash memory or other similar solid state memory device, or an array ofdevices, including devices in a storage area network or otherconfigurations. A computer program product can be tangibly embodied inan information carrier. The computer program product may also containinstructions that, when executed, perform one or more methods, such asthose described herein. The information carrier is a computer- ormachine-readable medium, such as the memory 1404, the storage device1406, memory on processor 1402, or a propagated signal.

The high speed controller 1408 manages bandwidth-intensive operationsfor the computing device 1400, while the low speed controller 1415manages lower bandwidth-intensive operations. Such allocation offunctions is exemplary only. In one implementation, the high-speedcontroller 1408 is coupled to memory 1404, display 1416 (e.g., through agraphics processor or accelerator), and to high-speed expansion ports1410, which may accept various expansion cards (not shown). In theimplementation, low-speed controller 1415 is coupled to storage device1406 and low-speed expansion port 1414. The low-speed expansion port,which may include various communication ports (e.g., USB, Bluetooth,Ethernet, or wireless Ethernet) may be coupled to one or moreinput/output devices, such as a keyboard, a pointing device, a scanner,an optical reader, a fluorescent signal detector, or a networking devicesuch as a switch or router, e.g., through a network adapter.

The computing device 1400 may be implemented in a number of differentforms, as shown in the figure. For example, it may be implemented as astandard server 1420, or multiple times in a group of such servers. Itmay also be implemented as part of a rack server system 1424. Inaddition, it may be implemented in a personal computer such as a laptopcomputer 1422. Alternatively, components from computing device 1400 maybe combined with other components in a mobile device (not shown), suchas device 1450. Each of such devices may contain one or more ofcomputing device 1400, 1450, and an entire system may be made up ofmultiple computing devices 1400, 1450 communicating with each other.

Computing device 1450 includes a processor 1452, memory 1464, aninput/output device such as a display 1454, a communication interface1466, and a transceiver 1468, among other components (e.g., a scanner,an optical reader, a fluorescent signal detector). The device 1450 mayalso be provided with a storage device, such as a microdrive or otherdevice, to provide additional storage. Each of the components 1450,1452, 1464, 1454, 1466, and 1468, are interconnected using variousbuses, and several of the components may be mounted on a commonmotherboard or in other manners as appropriate.

The processor 1452 can execute instructions within the computing device1450, including instructions stored in the memory 1464. The processormay be implemented as a chipset of chips that include separate andmultiple analog and digital processors. The processor may provide, forexample, for coordination of the other components of the device 1450,such as control of user interfaces, applications run by device 1450, andwireless communication by device 1450.

Processor 1452 may communicate with a user through control interface1458 and display interface 1456 coupled to a display 1454. The display1454 may be, for example, a TFT LCD (Thin-Film-Transistor Liquid CrystalDisplay) or an OLED (Organic Light Emitting Diode) display, or otherappropriate display technology. The display interface 1456 may compriseappropriate circuitry for driving the display 1454 to present graphicaland other information to a user. The control interface 1458 may receivecommands from a user and convert them for submission to the processor1452. In addition, an external interface 1462 may be provide incommunication with processor 1452, so as to enable near areacommunication of device 1450 with other devices. External interface 1462may provide, for example, for wired communication in someimplementations, or for wireless communication in other implementations,and multiple interfaces may also be used.

The memory 1464 stores information within the computing device 1450. Thememory 1464 can be implemented as one or more of a computer-readablemedium or media, a volatile memory unit or units, or a non-volatilememory unit or units. Expansion memory 1474 may also be provided andconnected to device 1450 through expansion interface 1472, which mayinclude, for example, a SIMM (Single In Line Memory Module) cardinterface. Such expansion memory 1474 may provide extra storage spacefor device 1450, or may also store applications or other information fordevice 1450. For example, expansion memory 1474 may include instructionsto carry out or supplement the processes described herein, and mayinclude secure information also. Thus, for example, expansion memory1474 may be provide as a security module for device 1450, and may beprogrammed with instructions that permit secure use of device 1450. Inaddition, secure applications may be provided via the SIMM cards, alongwith additional information, such as placing identifying information onthe SIMM card in a non-hackable manner.

The memory may include, for example, flash memory and/or NVRAM memory,as discussed below. In one implementation, a computer program product istangibly embodied in an information carrier. The computer programproduct contains instructions that, when executed, perform one or moremethods, such as those described herein. The information carrier is acomputer- or machine-readable medium, such as the memory 1464, expansionmemory 1474, memory on processor 1452, or a propagated signal that maybe received, for example, over transceiver 1468 or external interface1462.

Device 1450 may communicate wirelessly through communication interface1466, which may include digital signal processing circuitry wherenecessary. Communication interface 1466 may provide for communicationsunder various modes or protocols, such as GSM voice calls, SMS, EMS, orMMS messaging, CDMA, TDMA, PDC, WCDMA, CDMA2000, or GPRS, among others.Such communication may occur, for example, through radio-frequencytransceiver 1468. In addition, short-range communication may occur, suchas using a Bluetooth, WiFi, or other such transceiver (not shown). Inaddition, GPS (Global Positioning System) receiver module 1470 mayprovide additional navigation- and location-related wireless data todevice 1450, which may be used as appropriate by applications running ondevice 1450.

Device 1450 may also communicate audibly using audio codec 1460, whichmay receive spoken information from a user and convert it to usabledigital information. Audio codec 1460 may likewise generate audiblesound for a user, such as through a speaker, e.g., in a handset ofdevice 1450. Such sound may include sound from voice telephone calls,may include recorded sound (e.g., voice messages, music files, etc.) andmay also include sound generated by applications operating on device1450.

The computing device 1450 may be implemented in a number of differentforms, as shown in the figure. For example, it may be implemented as acellular telephone 1480. It may also be implemented as part of asmartphone 1482, personal digital assistant, or other similar mobiledevice.

Various implementations of the systems and techniques described hereincan be realized in digital electronic circuitry, integrated circuitry,specially designed ASICs (application specific integrated circuits),computer hardware, firmware, software, and/or combinations thereof.These various implementations can include implementation in one or morecomputer programs that are executable and/or interpretable on aprogrammable system including at least one programmable processor, whichmay be special or general purpose, coupled to receive data andinstructions from, and to transmit data and instructions to, a storagesystem, at least one input device, and at least one output device.

These computer programs (also known as programs, software, softwareapplications or code) include machine instructions for a programmableprocessor, and can be implemented in a high-level procedural and/orobject-oriented programming language, and/or in assembly/machinelanguage. As used herein, the terms “machine-readable medium” and“computer-readable medium” refer to any computer program product,apparatus and/or device (e.g., magnetic discs, optical disks, memory,and Programmable Logic Devices (PLDs)) used to provide machineinstructions and/or data to a programmable processor, including amachine-readable medium that receives machine instructions as amachine-readable signal. The term “machine-readable signal” refers toany signal used to provide machine instructions and/or data to aprogrammable processor.

To provide for interaction with a user, the systems and techniquesdescribed herein can be implemented on a computer having a displaydevice (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display)monitor) for displaying information to the user and a keyboard and apointing device (e.g., a mouse or a trackball) by which the user canprovide input to the computer. Other kinds of devices can be used toprovide for interaction with a user as well; for example, feedbackprovided to the user can be any form of sensory feedback (e.g., visualfeedback, auditory feedback, or tactile feedback); and input from theuser can be received in any form, including acoustic, speech, or tactileinput.

The systems and techniques described herein can be implemented in acomputing system that includes a back end component (e.g., as a dataserver), or that includes a middleware component (e.g., an applicationserver), or that includes a front end component (e.g., a client computerhaving a graphical user interface or a Web browser through which a usercan interact with an implementation of the systems and techniquesdescribed herein), or any combination of such back end, middleware, orfront end components. The components of the system can be interconnectedby any form or medium of digital data communication (e.g., acommunication network). Examples of communication networks include alocal area network (“LAN”), a wide area network (“WAN”), and theInternet.

The computing system can include clients and servers. A client andserver are generally remote from each other and typically interactthrough a communication network. The relationship of client and serverarises by virtue of computer programs running on the respectivecomputers and having a client-server relationship to each other.

In some cases, a system provided herein can be configured to include oneor more sample analyzers. A sample analyzer can be configured to producea plurality of signals about genomic DNA of a cancer cell. For example,a sample analyzer can produce signals that are capable of beinginterpreted in a manner that identifies the allelic imbalance status ofloci along a chromosome. In some cases, a sample analyzer can beconfigured to carry out one or more steps of a sequencing-based assayand can be configured to produce and/or capture signals from suchassays. In some cases, a computing system provided herein can beconfigured to include a computing device. In such cases, the computingdevice can be configured to receive signals from a sample analyzer.

The computing device can include computer-executable instructions or acomputer program (e.g., software) containing computer-executableinstructions for carrying out one or more of the methods or stepsdescribed herein. In some cases, such computer-executable instructionscan instruct a computing device to analyze signals from a sampleanalyzer, from another computing device, or from a sequencing-basedassay. The analysis of such signals can be carried out to determinegenotypes, allelic imbalance at certain loci, regions of allelicimbalance, the number of allelic imbalance regions, to determine thesize of allelic imbalance regions, to determine the number of allelicimbalance regions having a particular size or range of sizes, or todetermine a combination of these items.

In some cases, a system provided herein can include computer-executableinstructions or a computer program (e.g., software) containingcomputer-executable instructions for formatting an output providing anindication about copy number, allelic imbalance, LOH, or a combinationof these items.

In some cases, a system provided herein can include a pre-processingdevice configured to process a sample (e.g., cancer cells) such that asequencing-based assay can be performed. Examples of pre-processingdevices include, without limitation, devices configured to enrich cellpopulations for cancer cells as opposed to non-cancer cells, devicesconfigured to lyse cells and/or extract genomic nucleic acid, anddevices configured to enrich a sample for particular genomic DNAfragments.

Additional embodiments of the invention are as follows:

Embodiment 1

An in vitro method of detecting allelic imbalance status in a pluralityof genomic loci in a sample from a patient, comprising:

-   -   enriching a genomic DNA sample for DNA molecules each comprising        a locus of interest;    -   sequencing said DNA molecules to determine the genotype at each        such locus;    -   determining for each locus whether it has allelic imbalance.

Embodiment 2

An in vitro method of detecting LOH status in a plurality of genomicloci in a sample from a patient, comprising:

-   -   enriching a genomic DNA sample for DNA molecules each comprising        a locus of interest;    -   sequencing said DNA molecules to determine the genotype at each        such locus;    -   determining for each homozygous locus whether it is homozygous        due to LOH.

Embodiment 3

A system for determining allelic imbalance status in a plurality ofgenomic loci in a sample comprising:

-   -   a sample analyzer for (1) enriching a genomic DNA sample for DNA        molecules each comprising a locus of interest and (2) sequencing        said DNA molecules to produce a plurality of quantitative        signals about each such locus;    -   a computer program for analyzing said plurality of quantitative        signals to determine the genotype of each such locus in the        sample; and    -   a computer program for determining for each locus whether it has        allelic imbalance.

Embodiment 4

A system for determining LOH status in a plurality of genomic loci in asample comprising:

-   -   a sample analyzer for (1) enriching a genomic DNA sample for DNA        molecules each comprising a locus of interest and (2) sequencing        said DNA molecules to produce a plurality of quantitative        signals about each such locus;    -   a computer program means for analyzing said plurality of        quantitative signals to determine the genotype of each such        locus in the sample; and    -   a computer means for determining for each homozygous locus        whether it is homozygous due to LOH.

Embodiment 5

The method of either Embodiment 1 or Embodiment 2 or the system ofeither Embodiment 3 or Embodiment 4, wherein said plurality of genomicloci comprises at least 10, 50, 100, 1,000, 10,000, 50,000, 55,000,75,000, 100,000, 150,000, 200,000, 300,000, 400,000, 500,000, 750,000,1,000,000, or 2,000,000 or more loci.

Embodiment 6

The method or system of Embodiment 5, wherein said genomic loci areevenly spaced along the genome.

Embodiment 7

The method or system of Embodiment 6, wherein the genomic spacing ofsaid plurality of genomic loci is less than or equal to 50%, 40%, 30%,20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

Embodiment 8

The method of either Embodiment 1 or Embodiment 2 or the system ofeither Embodiment 3 or Embodiment 4, wherein said sample is aformalin-fixed, paraffin-embedded tissue sample.

Embodiment 9

The method or system of Embodiment 8, wherein said sample is a tumorsample extracted from the patient.

Examples

The process described here utilized an Agilent SureSelect Capture systemfollowed by IIlumina HiSeq sequencing, however any in solution or solidsupport based capture method and high throughput parallel sequencingplatform could be used.

The initial design selection process utilized the ^(˜)2.5 million SNPson the IIlumina Omni2.5 SNP array. This list of SNPs was chosen becauseit is the currently the largest list of SNPs from which there isgenotyping information available for multiple different populationgroups. All 2,448,785 SNP locations were input into the Agilent eArraySure Select Target Enrichment wizard for Single End Long Reads using thedefault settings. 1,353,042 passed the selection criteria and had baitsdesigned.

Then, 110,000 SNPs with high minor allele frequences and evenly coveringthe genome were selected. In the selection, SNPs in strong linkagedisequilibriom and SNPs with stong deviation from Hardy-Weinbergequilibrium were discarded.

Two preliminary library designs were constructed comprised of 55,000probes each targeting 55,000 different SNP locations. Testing wascarried out using a high quality normal DNA sample to check for evencapture of both alleles of every SNP. In addition, 4 FFPE samples werecaptured and used to select the most optimally performing probes. Welooked for probes that showed robust capture and even sequence depthwithout over or underrepresentation of sequence reads in the finalsequencing library.

The final capture probe library design was comprised of the 55,000optimal probes identified using the preliminary capture designs.

The results of measuring copy number and LOH using the above sequencingtechnique are shown in FIG. 2 (with FIG. 1 showing microarray analysison fresh frozen tissue as a comparison).

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. The mere mentioning of thepublications and patent applications does not necessarily constitute anadmission that they are prior art to the instant application.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. An in vitro method of detecting loss ofheterozygosity (LOH) status in a plurality of single nucleotidepolymorphism (SNP) genomic loci in a formalin-fixed paraffin-embeddedtissue sample from a patient, comprising: enriching a genomic DNA samplefor DNA molecules each comprising a SNP locus of interest, wherein thereis at least one SNP locus located on average every 5 Mb within eachchromosome that is examined; sequencing the DNA molecules to determinethe genotype at each such SNP locus; determining for each homozygous SNPlocus whether it is homozygous due to LOH.
 2. The method of claim 1,wherein there is at least one SNP locus located on average every 1 Mbwithin each chromosome that is examined.
 3. The method of claim 1,wherein the plurality of genomic SNP loci comprises at least 5,000 moreloci.
 4. The method of claim 1, wherein the plurality of genomic SNPloci comprises at least 10,000 more loci.
 5. The method of claim 1,wherein a SNP locus is determined to have allelic imbalance if the majorcopy proportion at the SNP locus is 0.51 or greater.
 6. The method ofclaim 1, wherein the SNP genomic loci are evenly spaced along thegenome.
 7. The method of claim 6, wherein the percentage differencebetween the distance between any two of the plurality of SNP genomicloci and the distance between any other two of the plurality of SNP lociis less than or equal to 20%.
 8. The method of claim 1, wherein thesample is a tumor sample extracted from the patient.
 9. The method ofclaim 8, wherein the tumor sample comprises at least 5%, or morecontamination with non-tumor cells.
 10. The method of claim 1, whereinthere is at least one single nucleotide polymorphism locus located onaverage every 500 kb within each chromosome.
 11. A system fordetermining LOH status in a plurality of SNP genomic loci in aformalin-fixed paraffin-embedded tissue sample, wherein the systemcomprises: a sample analyzer configured to enrich a genomic DNA samplefor DNA molecules, each test DNA molecule comprising a SNP locus ofinterest and wherein there is at least one SNP locus located on averageevery 5 Mb within each chromosome that is examined; sequencing the DNAmolecules to produce a plurality of quantitative signals for each suchSNP locus; a computer program that is configured to analyze theplurality of quantitative signals to determine the genotype of each suchSNP locus in the sample; and a computer program that is configured todetermine, for each homozygous SNP locus, whether the locus ishomozygous due to LOH.
 12. The system of claim 11, wherein there is atleast one SNP locus located on average every 1 Mb within each chromosomethat is examined.
 13. The system of claim 11, wherein the plurality ofgenomic SNP loci comprises at least 5,000 more loci.
 14. The system ofclaim 11, wherein the plurality of genomic SNP loci comprises at least10,000 more loci.
 15. The system of claim 11, wherein a SNP locus isdetermined to have allelic imbalance if the major copy proportion at theSNP locus is 0.51 or greater.
 16. The system of claim 11, wherein theSNP genomic loci are evenly spaced along the genome.
 17. The system ofclaim 16, wherein the percentage difference between the distance betweenany two of the plurality of SNP genomic loci and the distance betweenany other two of the plurality of SNP loci is less than or equal to 20%.18. The system of claim 11, wherein the sample is a tumor sampleextracted from the patient.
 19. The system of claim 18, wherein thetumor sample comprises at least 5%, or more contamination with non-tumorcells.
 20. The system of claim 11, wherein there is at least one singlenucleotide polymorphism locus located on average every 500 kb withineach chromosome.